**4. Application of PEF for microbial decontamination in wineries**

PEF treatments have been shown to cause microbial inactivation of vegetative cells of bacteria, yeast, and molds. Bacterial spores are resistant to PEF; nevertheless, since spores are not able to proliferate under acidic conditions, PEF represents a worthwhile alternative for the stabilization of acidic food such as must and wine. To implement PEF technology as a preservation method in wineries, it would be essential to determine the target microorganisms in every step of its application, and to conduct studies to prove that it ensures the level of microbial decontamination required to avoid spoilage. Finally, optimized PEF conditions should be applicable at an industrial scale without any negative effect on the appreciated quality properties of wine.

Several studies have demonstrated the potential of PEF for the inactivation of bacteria and yeast in must and wine. **Figure 2** shows the different winemaking steps in which the effectiveness of PEF for microbial decontamination and/or control of the microbial population in must or wine has been investigated. The main results obtained in those studies are described below.

#### **4.1 Application of PEF for decontamination of must**

PEF has proven highly effective in the inactivation of diverse microorganisms present in several kinds of fruit juice, including grape juice [21–23]. Reduction rates ranging from 2.0 to 4.0 log cycles were obtained by PEF (35 kV/cm, 1 ms) in must contaminated by a mixture of spoilage yeast and bacteria, such as *Saccharomyces cerevisiae, Kloeckera apiculata*, *Lactobacillus plantarum*, *Lactobacillus hilgardii*, and *Gluconobacter oxydans* [24]. In that study, the lethality of PEF was higher for yeast than for bacteria. Wu et al. achieved 4.0 log cycles of reduction in the natural spoilage flora of grape juice by applying a more intense PEF treatment (80 kV/cm, 40 μs) at 50°C that did not affect the juice's vitamin C content [25]. Further inactivation rates (up to 5.0 log cycles) were obtained when PEF was combined with certain antimicrobials such as lysozyme and nisin. Puértolas et al. established an optimum treatment of 186 kJ/kg at 29 kV/cm, reducing 99.9% of the spoilage flora of artificially contaminated must [26]. Moreover, PEF treatments have been shown

#### **Figure 2.**

*Steps of winemaking in which pulsed electric fields have potential application for microbial control and decontamination.*

to cause no significant changes in the physicochemical and nutritional properties of must, even when they are combined with mild temperatures (<50°C) [27, 28].

Studies in near-actual winemaking conditions have been conducted to evaluate the potential of PEF for replacing SO2 prior to alcoholic fermentation, with the objective of stabilizing the must and thus facilitating the growth of the culture starters. PEF treatments in must at 35 kV/cm for 1 ms was shown to be effective for controlling the microbial population before the inoculation of the yeast strains selected for alcoholic fermentation. The wines obtained after the alcoholic fermentation of PEF-treated must do not show any change in terms of their volatile profile, nor any modification of their characteristics after subsequent aging in bottles in comparison to wines added with SO2 [29, 30]. Alternatively, the use of non-*Saccharomyces* strains for alcoholic fermentation and for the improvement of the sensorial profile of neutral varieties is becoming a new trend in winemaking. Certain studies have confirmed that non-*Saccharomyces* yeasts implant themselves better in PEF-treated must [31, 32]. Consequently, higher levels of several specific metabolites of interest produced by non-*Saccharomyces* yeasts have been detected in wines obtained from PEF-treated musts.

Therefore, must stabilization by PEF is proving to be a good alternative for the reduction or elimination of the SO2 dose, thereby facilitating the implementation of selected *Saccharomyces* and non-*Saccharomyces* yeast starters for purposes of alcoholic fermentation.

#### **4.2 Application of PEF for wine decontamination after alcoholic fermentation**

Although *S. cerevisiae* strains are predominant in wine after alcoholic fermentation (AF), certain other non-*Saccharomyces* yeasts may persist due to their ethanol tolerance. Not only yeasts, but also LAB and AAB from grapes and even other microbes present in winery facilities or in the environment can contaminate

#### *Microbial Decontamination by Pulsed Electric Fields (PEF) in Winemaking DOI: http://dx.doi.org/10.5772/intechopen.101112*

the wine. Some wines are subjected to malolactic fermentation (MLF) after AF. Generally, starter cultures of LAB are added to the freshly fermented wine to ensure good implantation and prevent the proliferation of undesirable bacteria. The usual addition of SO2 prior to MLF can limit or hamper the implantation of the selected starters. PEF has thus been studied as a viable decontamination technique capable of reducing the competitive pressure exerted on MLF culture starters in freshly fermented wine.

González-Arenzana et al. tested the efficacy of PEF treatments in Tempranillo red wine at 17, 21 and 23 kV/cm (from 60 to 95 kJ/kg) in the inactivation of 25 different species of wine-associated microbiota [33]. Inactivation levels ranged from 1.70 to 3.04 log units for yeasts, from 1.01 to 4.16 for LAB, and from 0.64 to 4.94 for AAB. Similarly, Abca & Evrendilek investigated the effectivity of PEF treatments against a series of microbial strains suspended in red wine [34]. A PEF treatment at 31 kV/cm caused a reduction of more than 5.0 log cycles in the yeast population of *Saccharomyces cerevisiae*, *Hansenula anomala (Pichia anomala)*, and *Candida lipolytica*. Levels of inactivation of *Escherichia coli* and *Lactobacillus bulgaricus* with the same PEF treatment were 3.6 and 4.0 log cycles, respectively.

The application of PEF as an alternative to the addition of SO2 in sweet wines to prevent re-fermentation was investigated by Delsart et al. [35]. A PEF treatment (20 kV/cm, 320 kJ/kg) inactivated 3.0 and 4.0 log cycles for *Saccharomyces* and *non-Saccharomyces* strains, respectively. Although the addition of SO2 (250 mg/L) or the application of high-voltage electrical discharges (HVEDs) had a slightly greater lethal effect, PEF treatments caused less browning in the treated wines.

Attending to new consumer trends toward overall reduction of alcohol intake, wineries are producing low-alcohol wines [36]. Lower alcohol concentration might nevertheless lead to a higher risk of proliferation of spoiling or undesirable microorganisms in wine. PEF treatments (40 kV/cm, 250 μ) achieved inactivation levels up to 1.5 and 2.0 log cycles of LAB and yeasts in wines which had only 8.5% alcohol content [37].

Furthermore, a PEF treatment of 158 kJ/kg (33 kV/cm) has been validated as an improvement of the implementation of MLF starters in the production of four Tempranillo Rioja wines. The PEF-treated wines that were subjected to MLF preserved all their sensorial properties, as determined by sensory analysis through an expert panel [38].

*Brettanomyces spp.* is regarded as one of the most damaging and undesirable microorganisms in the wine industry due to its the high negative impact on the sensory properties of wines, even at very low concentrations. The capacity of PEF for the reduction of the population of this microorganism has been investigated by different authors. It has been observed that the lethal effect of PEF depends on the processing conditions, but differences in terms of PEF resistance among different strains have likewise been ascertained. Similar inactivation was achieved through a series of different combinations of electric field intensity and total specific energy in treatments applied under batch conditions. Inactivation of up to 4.0 log cycles was reported by applying 31 kV/cm and 150 kJ/kg [26] or 20 kV/cm and 320 kJ/kg [35]. Inactivation in the range of 2.5 to 3.0 log cycles was reported when the treatments were applied in continuous flow [33, 39].
